The amyloidogenic, protease-resistant isoform (PrP-res) of the host protein PrP is specific to scrapie and related transmissible spongiform encephalopathies (TSEs) and its accumulation in the CNS is important in the pathogenesis of these neurodegenerative diseases. Our major goals are to determine 1) the mechanism of PrP-res formation in scrapie- infected cells, 2) methods of preventing PrP-res accumulation, and 3) the relationship of PrP-res to the transmissible scrapie agent. Last year we reported studies of the kinetics and subcellular site of PrP-res formation in scrapie-infected neuroblastoma cells. Using this in vitro model, we have now identified potent inhibitors of the accumulation of PrP-res which are effective at concentrations as low as 1 ng/ml. These inhibitors include the amyloid-binding dye, Congo red, and several natural and synthetic sulfated glycan polyanions. Metabolic labeling studies indicated that Congo red has no effect on normal PrP metabolism and thus appears to selectively disrupt its conversion to PrP-res or destabilizes PrP-res once it is made. We have compared the inhibitory properties of a wide range of polyanions in an effort to define the structural requirements for good inhibitors of PrP-res accumulation. Such selective inhibitors should be valuable probes for studying the mechanism of PrP-res accumulation and the relationship of PrP-res to the transmissible scrapie agent. As an example of the latter application, we are now testing whether a decrease in the scrapie infectivity accompanies the reduction of PrP-res in Congo red-treated neuroblastoma cells. In vivo studies of the effects of Congo red on the development of scrapie in mice and hamsters are also underway. Because Congo red characteristically binds to all amyloids, including the a-peptide fibrils of Alzheimer's disease, we speculate that it may be capable of inhibiting the accumulation of a broad spectrum of amyloidogenic proteins in vivo and have therapeutic value in treating amyloidoses. Another approach we are taking to define the molecular mechanism of PrP-res formation is the comparison of the conformations of PrP-res and its normal isoform by infrared spectroscopy. The secondary structure analysis of PrP-res fibrils has been completed. We are continuing our refinement of both the infrared technique and a nondenaturing immunoaffinity purification of normal PrP so that we can complete the comparison.